The two major cellular components of the vasculature are the endothelial and smooth muscle cells. The endothelial cells form the lining of the inner surface of all blood vessels and constitute a nonthrombogenic interface between blood and tissue. In addition, endothelial cells are an important component for the development of new capillaries and blood vessels. Thus, endothelial cells proliferate during the angiogenesis, or neovascularization, associated with tumor growth and metastasis, as well as a variety of non-neoplastic diseases or disorders.
Various naturally occurring polypeptides reportedly induce the proliferation of endothelial cells. Among those polypeptides are the basic and acidic fibroblast growth factors (FGF), Burgess and Maciag, Annual Rev. Biochem., 58:575 (1989), platelet-derived endothelial cell growth factor (PD-ECGF), Ishikawa et al., Nature, 338:557 (1989), and vascular endothelial growth factor (VEGF), Leung et al., Science, 246:1306 (1989); Ferrara and Henzel, Biochem. Biophys. Res. Commun., 161:851 (1989); Tischer et al., Biochem. Biophys. Res. Commun., 165:1198 (1989); Ferrara et al., PCT Pat. Pub. No. WO 90/13649 (published Nov. 15, 1990).
VEGF has been reported as a key regulator of angiogenesis and vasculogenesis. Ferrara and Davis-Smyth (1997)Endocrine Rev. 18:4–25. Compared to other growth factors that contribute to the processes of vascular formation, VEGF is unique in its high specificity for endothelial cells. It is important not only for normal physiological processes such as wound healing, the female reproductive tract, bone/cartilage formation and embryonic formation, but also during the development of conditions or diseases that involve pathological angiogenesis, for example, tumor growth, age-related macular degeneration (AMD) and diabetic retinopathy. Ferrara and Davis-Smyth (1997), supra; Folkman J. (1995) Nature Med. 1:27–31; Pepper MS. (1997) Arterioscler Thromb. Vasc. Biol. 17:605–619.
In addition to being an angiogenic factor in angiogenesis and vasculogenensis, VEGF, as a pleiotropic growth factor, exhibits multiple biological effects in other physiological and pathological processes, such as endothelial cell survival, vessel permeability and vasodilation, monocyte chemotaxis and calcium influx. Ferrara and Davis-Smyth (1997), supra.
VEGF was first identified in media conditioned by bovine pituitary follicular or folliculostellate cells. Biochemical analyses indicate that bovine VEGF is a dimeric protein with an apparent molecular mass of approximately 45,000 Daltons and with an apparent mitogenic specificity for vascular endothelial cells. DNA encoding bovine VEGF was isolated by screening a cDNA library prepared from such cells, using oligonucleotides based on the amino-terminal amino acid sequence of the protein as hybridization probes.
Human VEGF was obtained by first screening a cDNA library prepared from human cells, using bovine VEGF cDNA as a hybridization probe. One cDNA identified thereby encodes a 165-amino acid protein having greater than 95% homology to bovine VEGF; this 165-amino acid protein is typically referred to as human VEGF (hVEGF) or VEGF165. The mitogenic activity of human VEGF was confirmed by expressing the human VEGF cDNA in mammalian host cells. Media conditioned by cells transfected with the human VEGF cDNA promoted the proliferation of capillary endothelial cells, whereas control cells did not. [See Leung et al., Science, 246:1306 (1989)].
Although a vascular endothelial cell growth factor could be isolated and purified from natural sources for subsequent therapeutic use, the relatively low concentrations of the protein in follicular cells and the high cost, both in terms of effort and expense, of recovering VEGF proved commercially unavailing. Accordingly, further efforts were undertaken to clone and express VEGF via recombinant DNA techniques. [See, e.g., Laboratory Investigation, 72:615 (1995), and the references cited therein].
VEGF is expressed in a variety of tissues as multiple homodimeric forms (121, 145, 165, 189, and 206 amino acids per monomer) resulting from alternative RNA splicing. VEGF121 is a soluble mitogen that does not bind heparin; the longer forms of VEGF bind heparin with progressively higher affinity. The heparin-binding forms of VEGF can be cleaved in the carboxy terminus by plasmin to release a diffusible form(s) of VEGF. Amino acid sequencing of the carboxy terminal peptide identified after plasmin cleavage is Arg110–Ala111. Amino terminal “core” protein, VEGF (1–110) isolated as a homodimer, binds neutralizing monoclonal antibodies (such as the antibodies referred to as 4.6.1 and 3.2E3.1.1) and soluble forms of FLT-1 and KDR receptors with similar affinity compared to the intact VEGF165 homodimer.
Certain VEGF-related molecules have also been identified. Ogawa et al. described a gene encoding a polypeptide (called VEGF-E) with about 25% amino acid identity to mammalian VEGF. The VEGF-E was identified in the genome of Orf virus (NZ-7 strain), a parapoxvirus that affects sheep and goats and occasionally, humans, to generate lesions with angiogenesis. The investigators conducted a cell proliferation assay and reported that VEGF-E stimulated the growth of human umbilical vein endothelial cells as well as rat liver sinusoidal endothelial cells to almost the same degree as human VEGF. Binding studies were also reported. A competition experiment was conducted by incubating cells that overexpressed either the KDR receptor or the FLT-1 receptor with fixed amounts of 125I-labeled human VEGF or VEGF-E and then adding increasing amounts of unlabeled human VEGF or VEGF-E. The investigators reported that VEGF-E selectively bound KDR receptor as compared to FLT-1. [Ogawa et al. J. Biological Chem. 273:31273–31281 (1998)].
Meyer et al., EMBO J., 18:363–374 (1999), have also identified a member of the VEGF family which is referred to as VEGF-E. The VEGF-E molecule reported by Meyer et al. was identified in the genome of Orf virus strain D1701. In vitro, the VEGF-E was found to stimulate release of tissue factor and stimulate proliferation of vascular endothelial cells. In a rabbit in vivo model, the VEGF-E stimulated angiogenesis in the rabbit cornea. Analysis of the binding properties of the VEGF-E molecule reported by Meyer et al., in certain assays revealed the molecule selectively bound to the KDR receptor as compared to the FLT-1 receptor.
Olofsson et al., Proc. Natl. Acad. Sci., 95:11709–11714 (1998) report that a protein referred to as “VEGF-B” selectively binds FLT-1. The investigators disclose a mutagenesis experiment wherein the Asp63, Asp64, and Glu67 residues in VEGF-B were mutated to alanine residues. Analysis of the binding properties of the mutated form of VEGF-B revealed that the mutant protein exhibited a reduced affinity to FLT-1.
VEGF contains two sites that are responsible respectively for binding to the KDR (kinase domain region) and FLT-1 (FMS-like tyrosine kinase) receptors. These receptors are believed to exist primarily on endothelial (vascular) cells. Recently, Soker et al. identified another VEGF receptor that has a sequence identical to neuropilin. Soker et al., Cell, 92:735–745 (1998). This receptor bound to VEGF165 and placental growth factor-2 (PLGF-2) but not to VEGF121. Soker et al., supra; Migdal et al., J. Biol. Chem. 273:22272–22278 (1998).
VEGF production increases in cells that become oxygen-depleted as a result of, for example, trauma and the like, thereby allowing VEGF to bind to the respective receptors to trigger the signaling pathways that give rise to a biological response. For example, the binding of VEGF to such receptors may lead to increased vascular permeability, causing cells to divide and expand to form new vascular pathways—i.e., vasculogenesis and angiogenesis. [See, e.g., Malavaud et al., Cardiovascular Research, 36:276–281 (1997)]. It is reported that VEGF-induced signaling through the KDR receptor is responsible for the mitogenic effects of VEGF and possibly, to a large extent, the angiogenic activity of VEGF. [Waltenberger et al., J. Biol. Chem., 269:26988–26995 (1994)]. The biological role(s) of FLT-1, however, is less well understood.
The sites or regions of the VEGF protein involved in receptor binding have been identified and found to be proximately located. [See, Weismann et al., Cell, 28:695–704 (1997); Muller et al., Proc. Natl. Acad. Sci., 94:7192–7197 (1997); Muller et al., Structure, 5:1325–1338 (1997); Fuh et al., J. Biol. Chem., 273:11197–11204 (1998)]. The KDR receptor has been found to bind VEGF predominantly through the sites on a loop which contains arginine (Arg or R) at position 82 of VEGF, lysine (Lys or K) at position 84, and histidine (His or H) at position 86. The FLT-1 receptor has been found to bind VEGF predominantly through the sites on a loop which contains aspartic acid (Asp or D) at position 63, glutamic acid (Glu or E) at position 64, and glutamic acid (Glu or E) at position 67. [Keyt et al., J. Biol. Chem., 271:5638–5646 (1996)]. Based on these findings, the wild type VEGF protein has been used as the starting point for introduction of mutations in specific receptor-binding sites, in attempts to create VEGF variants selectively bind to one receptor such as KDR. Keyt et al., supra; Shen et al. (1998) J. Biol. Chem. 273:29979–29985. The resulting VEGF variants showed moderate receptor selectivity. More recently, based on the crystal structure of VEGF and functional mapping of the KDR binding site of VEGF, it has further been found that VEGF engages KDR receptors using two symmetrical binding sites located at opposite ends of the molecule. Each site is composed of two “hot spots” for binding that consist of residues from both subunits of the VEGF homodimer. Muller et al., Structure, 5:1325–1338 (1997). Two of these binding determinants are located within the dominant hot spot on a short, 3-stranded beta-sheet that is conserved in transforming growth factor beta2 (TGF-beta) and platelet-derived growth factor (PDGF).
Recent studies report that endothelium-derived nitric oxide (NO) and endothelial NO synthase (eNOS) may play an important role in various VEGF-induced activities. NO is believed to be an important mediator of endothelial function and a regulator of vascular homeostasis, platelet aggregation, and angiogenesis. Busse and Flemming (1996) J. Vasc. Res. 33:181–194. NO is produced from conversion of L-arginine to citrulline by NO-synthase, an enzyme which consists of 3 isoforms denominated endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS). Nathan and Xie (1994) J. Biol. Chem. 269:13725–13728. VEGF has been shown to induce rapid release of NO from rabbit, pig, bovine and human vascular endothelial cells. (see, e.g., vanderZee et al., Circulation, 95:1030–1037 (1997); Parenti et al., J. Biol. Chem., 273:4220–4226 (1998); Morbidelli et al., Am. J. Physiol., 270:H411–H415 (1996); Papapetropoulis et al., J. Clin. Invest., 100:3131–3139 (1997); Kroll et al., Biochem. Biophys. Res. Comm., 252:743–746 (1998); Hood et al., Am. J. Phys., 274:H1054–H1058 (1998)). In an in vitro assay, VEGF was found to stimulate human endothelial cells to grow in a NO-dependent manner and promote the NO-dependent formation of vessel-like structures in the 3-D collagen gel model (Papapetropoulis et al., supra). Conversely, eNOS inhibitors were reported to inhibit VEGF-induced mitogenic and angiogenic effects. (Papapetropoulis et al., supra). In certain in vivo studies, inactivation of eNOS expression impaired VEGF-induced angiogenesis in an eNOS knockout mouse model. (Murohara et al., J. Clin. Invest., 101:2567–2578 (1998); Ziche et al., J. Clin. Invest., 99:2625–2634 (1997); Rudic et al., J. Clin. Invest., 101:731–736 (1998)). Recently, Liu et al., J. Biol. Chem., 274:15781–15785 (1999) reported that VEGF down-regulated an endogenous inhibitor of eNOS called Caveolin.
It has been shown that VEGF induces vasodilation in vitro in a dose-dependant fashion and produces transient tachicardia, hypotension and a decrease in cardiac output when injected intravenously in conscious, instrumented rats. Yang et al., J. Cardiovasc. Pharmacol., 27:838–844 (1996) Such acute hemodynamic effects appear to be caused by a decrease in venous return, mediated primarily by endothelial cell-derived NO. Yang et al., supra; Hariawala et al., J. Surg. Res., 63:77–82 (1996). Wu et al., Am. J. Physiol., 271:H2735–H2739 (1996) describe that topical application of VEGF resulted in a transient and dose-dependent increase in albumin permeability in isolated coronary venules.
Inhibition of eNOS by L-NAME, in vivo, has been reported to result in an increase in mean arterial pressure (MAP) (Yang et.al., 1996 Cardiovasc. Pharmacol. 27:838–844; Sase et.al., 1997 Trends J. Cardiovasc. Med. 7:28–37) and a decrease in angiogenesis (Papapetropoulos et.al., 1997 J.Clin. Invest. 100:3131–3139). Genetically engineered mice lacking the eNOS gene showed impaired endothelium-dependent vasodilation, angiogenesis, and hypertension (Murohara et.al., 1998 J.Clin. Invest. 101:2567–2578; Yang et. Al., 1996, supra), and over-expression of eNOS in mice by gene transfer was shown to increase nitric oxide production and significantly attenuated MAP and neointima formation (Sase et. al., supra; Drummond and Harrison, 1998, J. Clin. Invest. 102:2033–2044; Ohashi et.al., 1998 J. Clin. Invest. 102:2061–2071).
Although there have been a number of studies on the involvement of VEGF in short term release of NO and eNOS regulation, the chronic effect of VEGF on eNOS expression and/or activity has not been documented. Moreover, biological effects of sustained release of nitric oxide in treating or preventing vascular diseases in vivo have not been clearly demonstrated to date. This is due, in large part, to the fact that mammals can rapidly develop tolerance to certain agents exogenously administered as nitric oxide donors, making supplementation of nitric oxide difficult. (Drummond and Harrison, 1998, J. Clin. Invest. 102:2033–2044).
Up-regulation of eNOS expression by physiological or pharmacological approaches may provide a useful therapeutic approach to the treatment of diseases associated with endothelial cell dysfunction, for example, by increasing the production and sustained release of endogenous NO.